The ultimate goal of this project is to identify and analyze structural features of 2 human heat shock mRNAs that determine their high translational efficiency and different half lives. The metabolism and translational efficiency of mRNAs encoding the four major heat shock proteins and selected control proteins will be compared during hyperthermia in HeLa cells. Homologous cloned cDNA sequences and subcloned genomic DNA fragments will be used as hybridization probes. Further experiments will focus on the genes encoding the 70 Kd and 27 Kd heat shock polypeptides. The mRNA encoding the 70 Kd heat shock protein has a markedly shorter half-life during hyperthermia than that encoding the 27 Kd heat shock protein. Both mRNAs show high translational efficiencies. Structural genes for the human 70 Kd heat shock protein will be isolated from a genomic library. Two members of the 27 kd gene family are already available. Isolated genes and flanking DNA sequences will be introduced into mammalian cells and tested for heat inducible expression. Site specific in vitro mutagenesis will then be used to introduce alterations in different regions of the genes. The effect of various modifications on the ribosome binding efficiency and stability of transcripts will be measured in vivo. Identification of structural elements important for mRNA function is basic to an understanding of cytoplasmic control of gene expression. These studies should also increase our understanding of cellular processes which have been shown to be important for the development of thermotolerance, and which may also underlie the disruptive effects of hyperthermia on embryonic development, or the beneficial effects of hyperthermic treatment in cancer therapy. (G)